Anti il 6 antibody

For immunohistochemistry examination, kidney sections were immunostained using immunoperoxidase technique with Vector ABC kit (Vector Laboratories). Briefly, sections (3μm thick) were blocked with 3% BSA for 30 min at room temperature, and incubated with primary antibody overnight at 4°C. The primary antibodies used were anti-NF-κB p65 antibody (Santa Cruz Biotechnology mouse monoclonal, 1:200), anti-IL-6 antibody (Santa Cruz Biotechnology rabbit polyclonal, 1:200) and anti-ICAM-1 antibody (Santa Cruz Biotechnology mouse monoclonal, 1:200). Sections were then washed and incubated with biotinylated secondary antibodies for 60 min at room temperature. Biotin was identified and visualized with 3,3’-diaminobenzidine (DAB) solution. As a negative control, the primary antibody was replaced with nonimmune IgG, and no staining occurred. Counterstaining was then performed before examination under a light microscope. Random 100 glomeruli from each renal specimen were observed, and images were then analysed with Image Pro Plus 6.0 edition (Media Cybernetics) for the determination of immunostained area. The percentage of the stained area was calculated as the ratio of suitable binary thresholded image and the total field area.

For western blotting, 40ug of protein was electrophoresed on a 10% SDS-PAGE gel. Proteins were transferred onto a polyvinylidene difluoride membrane, and the membranes were hybridized in blocking buffer at 4 °C overnight with mouse monoclonal anti-CD68 antibody (1:1000, Cell Signaling Technology), rabbit polyclonal anti-TNF-α antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IL-6 antibody (1:500, Santa Cruz Biotechnology, USA), β-actin (1:500, Santa Cruz Biotechnology, USA), mouse monoclonal anti-fibronection antibody (1:1000, Cell Signaling Technology), mouse monoclonal anti-Type IV collagen antibody (1:1000, Cell Signaling Technology), mouse monoclonal anti-laminin antibody (1:1000, Cell Signaling Technology). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 60 min at room temperature and visualized using enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA). The relative amount of the proteins was then analyzed using Image J analysis software version 1.38e (NIH, Bethesda, MD) and normalized against their respective controls.

The middle segments of the small intestines were fixed, embedded, and sectioned as Swiss rolls for further immunohistochemical examination with the avidin–biotin complex immunoperoxidase technique. Polyclonal goat anti-COX-2 and anti-IL-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 200× dilution. As the secondary antibody, biotinylated anti-goat IgG, absorbed with horse serum (Vector Laboratories, Burlingame, CA) were employed at 200× dilution. Staining was performed using avidin–biotin reagents (Vectastain ABC reagents; Vector Laboratories), 3,3'-diaminobenzidine and hydrogen peroxide, and the sections were counterstained with hematoxylin to facilitate orientation. As a negative control, consecutive sections were immunostained without exposure to the primary antibody.

The tissues and cells were washed twice using ice-cold PBS and lysed in lysis buffer. Lysates were separated by sodium dodecyl sulfate-polyacrylamide (10%−12%) gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were washed with Tris-buffered saline/Tween-20 (TBST) and blocked in 5% skim milk powder dissolved in TBST for three hours, followed by the incubation with the respective primary antibody at dilutions according to the supplier's instructions.
The membranes were examined using an anti-NF-κB p65 antibody (1:1,000, Cell Signaling Technology), anti-heme oxygenase-1 (HO-1) antibody (1:1,000, Abcam), anti-Nrf2 antibody (1:1,000, Cell Signaling Technology), anti-Bax antibody (1:500, Santa Cruz Biotechnology), anti-cleaved caspase-3 antibody (1:500, Santa Cruz Biotechnology), anti-IL-6 antibody (1:500, Santa Cruz Biotechnology), anti-TNFα antibody (1:500, Santa Cruz Biotechnology), anti-nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) antibody (1:500, Santa Cruz Biotechnology), or anti-NQO1 antibody (1:500, Abcam). Following conjugation to horseradish peroxidase, the corresponding immunoglobulin G secondary antibody (1:1,000, Beyotime Biotechnology) was used to detect the primary antibodies. Enhanced chemiluminescence (Pierce, MA, USA) was used to visualize the bands.